GSM7838942: S2_Input_UN_WT; Mus musculus; ChIP-Seq
CH12F3 cells were either unstimulated or stimulated with a cocktail consisting of recombinant human TGFβ1 (R&D systems, 240-B, 1 ng/mL), recombinant mouse IL4 (Preprotech, 214-14, 10 ng/mL), and functional grade purified α-mouse CD40 (eBiosciences, 16-0401, 1 ug/mL) for 72 hours to induce IgA class-switching. Cells were fixed with 1% formaldehyde in the culture media for 10 minutes at room temperature, followed by addition of 0.125M of glycine to stop fixation. Nuclei were extracted with 5 minutes lysis in 0.25% Triton buffer, followed by 30 minutes in 200mM NaCl buffer. Nuclei were resuspended in sonication buffer and sonicated for twelve cycles of 30 seconds with a digital sonifier (Branson Ultrasonics) at 80%, with 30 seconds rest in cooled circulating water. Beads immunocomplexes were prepared overnight by conjugating 40μL of Dynabeads Protein G (Invitrogen, Life Technologies) with antibodies: anti-BAP1 (Cell Signaling Technology, D1W9B, 52.8 μg), anti-FLAG M2 (Sigma, F1804, 5 μg) or anti-H2AK119Ub (Cell Signaling Technology, D27C4, 5 μg). Immunoprecipitation was performed by overnight incubation of antibody-bead matrices with sheared chromatin from the equivalent of 5x106 cells. For BAP1-FLAG ChIP, 6 washes were performed with medium-stringency buffers, while 4 low-stringency washes were used for histone ChIP . Samples were de-crosslinked by overnight incubation at 65̊C in 1% SDS buffer. Following RNaseA and Proteinase K enzymatic treatments, ChIP DNA was purified using Qiaquick PCR Cleanup kit (Qiagen). DNA yields and quality were assessed using Bioanalyzer (Agilent). Library preparation were performed using the KAPA HyperPrep Kit. Sequencing was assessed using Novaseq 6000.